The simplicity and versatility of hydrazone crosslinking has made it a strategy of choice for the conjugation of bioactive molecules. However, the labile nature of hydrazone linkages and reversibility of this coupling reaction restricts its full potential. Based on the fundamental understanding of hydrazone stability, this problem is circumvented by resonance‐stabilization of a developing N2 positive charge in a hydrazone bond. A novel chemistry is presented to develop a resilient hydrazone bond that is stable and non‐ reversible under physiological conditions. A carbodihydrazide (CDH) type hydrazide derivative of the biomolecule forms intrinsically stabilized hydrazone‐linkages that are nearly 15‐fold more stable at pH 5 than conventional hydrazone. This chemoselective coupling reaction is catalyst‐free, instantaneous, and virtually non‐cleavable under physiological conditions, therefore can serve as a catalyst‐free alternative to click chemistry. This novel crosslinking reaction is used to tailor a hyaluronan hydrogel, which delivered exceptional hydrolytic stability, mechanical properties, low swelling, and controlled enzymatic degradation. These desired characteristics are achieved without increasing the chemical crosslinking. The in vivo evaluation of this hydrogel revealed neo‐bone with highly ordered collagen matrix mimicking natural bone regeneration. The proximity ligation assay or PLA is used to detect blood vessels, which highlighted the quality of engineered tissue.
Bone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs) dermatan sulfate (DS) or heparin (HP), prior to loading it into a hyaluronic acid (HA) hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG.
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